Sex determination was a founding topic ofC. elegansresearch. After three decades of
research, a complex signal transduction pathway with multiple layers of regulation has been elucidated. This pathway
links karyotype to phenotype by coordinating the
development of sexually dimorphic tissues. In this article, this pathway is placed in two broader contexts. The
first is that of nematodes and animals in general. The
important role ofC. elegansstudies in revealing the first universally conserved
component of metazoan sex determination is discussed, as is the role of cooption of genes into the sex determination
and dosage compensation pathways. The second context is
that of a subset of more closely related species, with emphasis on other members of the genusCaenorhabditis.Studies reviewed here have determined the gene-level conservation
of the known pathway and the relative rates of molecular evolution in conserved components, and made substantial
progress in the manipulation of gene activity in
non-elegansspecies. Special attention is paid to the origins of hermaphroditism, which evolved from gonochorism through germline-specific
changes in sex determination. Recent
studies suggest that the most rapidly evolving aspects of sex determination are germline functions related to
evolutionary shifts in mating systems, while somatic sex
determination is relatively conservative. From all of these studies, a picture emerges in whichC. elegansutilizes an intriguing mixture of general and species-specific genes
and regulatory mechanisms.

1. Introduction

Sex determination was one of the first aspects of C. elegans biology to be
systematically characterized with genetic analysis (Hodgkin and Brenner, 1977), and the depth of this analysis has made it a major topic in developmental
biology. It is both fair and interesting to ask how general the C. elegans model is
likely to be, both for other nematodes as well as for animals in general. As nematodes
employ many different reproductive strategies, often related to parasitic life histories
(Anderson, 2000; Chitwood and Chitwood, 1950), one might expect similarly
variable sex determination mechanisms. However, comparative studies of sex determination
based on the C. elgans model have revealed both rapidly evolving and surprisingly well
conserved features. This mixture of old and new (or slow and fast), along with its
obvious relevance to evolution, ecology, and applied fields like agricultural and
medical parasitology, has made the evolution of nematode sex determination an
increasingly active research area.

2. Genetic and environmental sex determination in nematodes

In 1949, Nigon demonstrated that C. elegans used an XX/XO sex chromosome
system, and the importance of the X:autosome ratio was clarified by Madl & Herman
(1979). The XX/XO system is widespread across the Nematoda, and may be
ancestral (Bull, 1981; Bull, 1983). However, some groups have apparently
abandoned genetic sex determination (GSD) in favor of environmental sex determination
(ESD).

The best characterized example of nematode ESD is in the arthropod parasitic
Mermithidae. In lightly infected hosts, exclusively female worms are born, while in
heavily parasitized hosts only males are produced. At intermediate parasite densities
mixed populations are produced (Christie, 1929; Harlos et al., 1980).
Culture of mermithids in axenic liquid culture also produces only females, suggesting
that the concentration of a limiting host factor may be an important cue for male
development (Petersen, 1985). Whether the environmental factors that
dictate sex impinge upon a signal transduction pathway resembling that mediating C
elegans sex determination is unknown, but just such a connection between nutrition and
sex determination has recently been found in C. elegans itself (Prahlad et al., 2003). Among the small number of nematode fossil specimens that exist are
mermithids emerging from their insect hosts, preserved in 40 million year old Baltic
amber (Poinar, 2002). Their striking similarity to extant
insect-parasitic species (Figure 1) suggests a minimum age for the origin of ESD in this
group, although it is possible that ancient mermithids used GSD.

ESD also occurs in the vertebrate parasite Strongyloides, which like C. elegans is in
the order Rhabditida. As with most parasitic nematodes, a dauer-like alternative L3 form
serves as the infective stage. Once inside a host, infective worms all mature as females
and reproduce by mitotic parthenogenesis (Anderson, 2000). Parthenogenetic progeny
that remain in the host develop into apomictic females, like their mothers. However,
when L1 larvae are passed from the host in feces, they can either develop into infective
L3 female larvae (the homogonic path) or initiate a free-living male/female generation
(the heterogonic phase) that is similar to Caenorhabditis. Heterogonic development
produces males and females from genetically identical L1 larvae, indicating that sex is
either stochastically or environmentally determined. This again resembles the sexual
plasticity of C. elegans larvae recently described by Prahlad et al. (2003), in which a chemical cue induces specific loss of the paternal X chromosome in
L1 larvae. It would be interesting to see if these phenomena share a common mechanism.

Figure 1. Ancient and extant mermithid nematodes emerging from their insect hosts. A. Juvenile worm of the genus Heydenius emerging from a winged male ant of the genus
Prenolepis. The specimen is preserved in Baltic amber approximately 40 million years
old. The scale bar represents 1.2 mm. Photograph reproduced from Poinar (2002) with
permission from the author and Cambridge Univ. Press. B. Juvenile mermithid emerging
from an extant mosquito larva. Photo used with permission of the Univ. of
Nebraska, Lincoln Dept. of Entomology.

Commitment to heterogonic development in Strongyloides requires amphid neurons
homologous to those that govern dauer formation in C. elegans (Ashton et al., 1998), indicating that the alternate reproductive modes are also environmentally
controlled. Males are required for female fertility in the heterogonic phase, but
apparently the male sperm serves only to activate embryogenesis. Diploidy is thought to
be maintained by fusion of the post-meiotic oocyte pronucleus with an oogenic polar body
(Anderson, 2000). The heterogonic phase is therefore pseudo-sexual, and presumably
evolved from a truly sexual ancestor. Given that parasites often rely on genetic
diversity for successful host invasion, this represents an unusual case that deserves
further investigation.

3. Deep conservation and cooption in the evolution of nematode sex determination

Although C. elegans, Drosophila, and mammals (the three most-studied systems) all use
GSD, the signal transduction pathways that convert the genetic difference between sex
into dimorphic fates are not at all similar (Cline and Meyer, 1996).
However, recently a conserved component has been discovered: the Doublesex/mab-3 (DM)
family of transcriptional regulators (Figure 2, Raymond et al., 1998; Yi
and Zarkower, 1999). Although mab-3 functions in specifying a limited
number of male cell fates in C. elegans, the role of Dsx in Drosophila and of DM
proteins in some vertebrates (Matsuda et al., 2002) indicate they can
function more globally. The association of DM family member expression with sexual
development in cnidarians (Miller et al., 2003) further suggests that
this role evolved prior to the Cambrian, over 500 million years ago. It is reasonable to
imagine that the first metazoans had sexual differentiation controlled in part by a DM
progenitor, and that the diversity of extant sex determination mechanisms represents
extreme divergence in different lineages. In this sense, then, sex determination itself
is a homologous developmental process, even though the genes that control it are in most
cases not themselves homologous.

How did the nematode system evolve? One interesting possibility is that much of it
represents a highly modified form of the hedgehog (hh) pathway. hh signaling controls
many key patterning and cell fate decisions in metazoan development, but is
conspicuously absent from C. elegans, as judged by the lack of bona fide hh and
smoothened homologs in the genome. However, the TRA-2 transmembrane receptor is
topologically similar to patched, and the pathway culminates in the regulation of a
Cubitus interruptus/Gli-like transcription factor, TRA-1. Perhaps in the distant past,
as nematode development became more mosaic and increasingly dependent upon cell lineage,
hh signaling became dispensable for other aspects of development and was coopted into
sex determination. Given the unusually high rates of sequence evolution exhibited by
many sex determination genes, it is plausible that some “novel” proteins in the pathway
may have at one time been recognizably similar to other hh-related homologs. The
isolation of her-1 (Streit et al., 1999) and tra-1 (Pires-daSilva and
Sommer, 2004) homologs from Brugia malayi (from the order Spirurida) and
Pristionchus pacificus (Rhabditida, Neodiplogasteridae), respectively, suggests that
this putative cooption happened early in the diversification of nematodes. In the case
of Ppa-tra-1, multiple mutant alleles provide conclusive proof of a conserved
female-promoting role.

Figure 2. Variation in model system sex determination. Though mammals, Drosophila, and C. elegans all use GSD, they interpret their sex
chromosome content through distinct signal transduction pathways. However, all three
eventually converge on a DM family member whose expression is associated with male
development. DrosophilaDsx is unusual for DM genes in also having an important role in
female development (through a female-specific splice variant).

There are two other likely cases of cooption of genes into Caenorhabditis sex
determination, enabled through pleiotropic function of a single gene or the divergence
of gene duplicates. First, the recent structural characterization of XOL-1 suggests that
it is related to small molecule metabolic kinases (Luz et al., 2003).
Whether XOL-1 actually acts as an enzyme is not yet clear, but the fact that it is the
least conserved protein in the entire sex determination pathway suggests it may not. The
other case is comprised of the dosage compensation complex genes. The products of mix-1,
dpy-26, dpy-27, and dpy-28 are clearly related to the 13S condensin complex that
mediates chromatin condensation in vertebrates (Meyer, 2000). MIX-1 has
been shown to play a direct role in both processes (Hagstrom et al., 2002), while dpy-26, dpy-27 and dpy-28 are replaced in cell division by products of
the paralogs C29E4.2 (Hagstrom and Meyer, pers. comm.), smc-4 (Hagstrom et al., 2002), and hcp-6 (Chan et al., 2004), respectively.

In 1995, Wilkins proposed that the many layers of negative regulation found in
nematode and Drosophila sex determination represent the accumulation of successive
upstream repressors, the so-called “bottom up” hypothesis”. The
conservation of tra-1 as a key female-promoting gene beyond the family Rhabditidae is
consistent with this idea. However, if the signal transduction pathway used in
Caenorhabditis sex determination is a diverged form of the hedgehog pathway, then one
might predict that the entire “cassette” from her-1 to tra-1 came into the pathway at
once, and thus will not be found in partial form in any species.

A more general test of the bottom-up idea will require examination of the more
upstream components of the pathway, such as the X chromosome counting factors fox-1 and
sex-1, in both close and distant relatives of C. elegans. Although the XX/XO system
seems to be widespread across the Nematoda, this does not mean that X dosage is measured
by homologous genes in all species. Although clear C. briggsaefox-1 and sex-1 homologs
exist, their functions remain untested. As of early 2005 an effort to sequence the
Pristionchus pacificus genome is underway, which should soon enable a search for these
genes (or their equivalents) in a second nematode family. Recently, Hodgkin (2002) demonstrated that C. elegans can be manipulated genetically such that any of
the core sex-determining genes (all autosomal), an extrachromosomal array, temperature,
or even a suppressor tRNA locus can substitute for the X:A ratio as the primary
determinant of sex. This impressive feat suggests that nature also has a large number of
options at its disposal for optimization of reproductive strategy. It will be of great
interest to see to what extent these options have, in fact, been used during nematode
evolution.

Homologs of C. elegans sex determination genes have been isolated from several
Caenorhabditis species, including the undescribed male/female species CB5161 (Stothard
et al., 2002), the male/female C. remanei (Chen et al., 2001; Haag and Kimble, 2000; Haag et al., 2002), and
the non-rhabditids Pristionchus pacificus (Pires-daSilva and Sommer, 2004) and Brugia malayi (Streit et al., 1999). However, the nearly
complete genome sequence assembly of C. briggsae (Stein et al., 2003)
makes it uniquely suited to assess molecular conservation across the entire C. elegans
pathway. The focus below is therefore on C. briggsae, but we shall return to the results
of functional studies in the other species in a later section.

Genes are arranged by where they act in the pathway, with sex chromosome counting elements at the top and tissue-specific
targets of TRA-1 at the bottom. Percent identities are based on published figures when available, on BLAST 2.0 alignments of predicted genes
from WormBase when alignable over at least 90% of their full length (B), or on whole alignments using the Needleman & Wunsch algorithm as implemented in the Gap program of the GCG Wisconsin Package (G). Genes that have not yet been cloned,
or whose products are involved in dosage compensation but not sex determination, are omitted here for simplicity. The median
amino acid identity across the entire set of C. elegans-C. briggsae orthologs is 80% (Stein and others, 2003). P, based on partial sequence; TF, transcription factor; RRM, RNA recognition motif.

Even well-conserved proteins can rapidly change their role (or lack thereof) in sex
determination, as has been found in dipteran insects (reviewed by Graham et al., 2003). Evidence for similar change in nematodes is discussed below. In
contrast, some proteins exhibiting rapid sequence change participate in conserved
protein-protein interactions with a consistent role in sex determination. For example,
the TRA-1/TRA-2c interaction is conserved in C. briggsae, and this interaction is
species-specific (Wang and Kimble, 2001). A more extreme case of
molecular coevolution exists between TRA-2c and FEM-3. The FEM-3 binding domain of
TRA-2c is conspicuously hyperdivergent (Haag and Kimble, 2000; Kuwabara,
1996), yet mediates a strong but species-specific interaction with the
similarly divergent FEM-3 in C. elegans, C. briggsae, and C. remanei (Haag et al., 2002). Why and how crucial protein-protein interactions mediating sex
determination are continually reinvented at the primary sequence level are interesting
questions that will require the integration of structural biology, genetics, and ecology
to be fully answered.

5. Two cases of species-specific genes

Though most C. elegans sex determination genes have a single C. briggsae ortholog,
there are two exceptions. fog-2 is clearly the result of a recent, elegans-specific
tandem duplication (Clifford et al., 2000). FOG-2 differs little from the
protein encoded by its nearest neighbor and most similar paralog, FTR-1, over its
N-terminal 80%, but has a distinct C-terminus. This divergent region is necessary and
sufficient for GLD-1 binding, and may be derived from the ancestral domain in part via a
frame-shift mutation (Nayak et al., 2005). fog-2 likely reveals the
footprints of a key event in the evolution of hermaphroditism in the C. elegans lineage.
This will be discussed at greater length below.

The second example of species-specific sex determination genes is that of fbf-1 and
fbf-2. These two Puf family genes are also the product of a recent tandem duplication,
and encode proteins that are 91% identical (Zhang et al., 1997). Three C.
briggsae genes, themselves products of recent lineage-specific duplication events, are
more closely related to the fbfs than to any other C. briggsae family members (Lamont et
al., 2004, A. Doty, unpublished data). However, the expectation that any
of these three C. briggsae genes perform the function of fbf in repressing fem-3 is
complicated by the lack of Cb-fem-3(RNAi) germline feminization (Haag et al., 2002). It may be that these fbf-related C. briggsae Puf proteins regulate Cb-fem-3,
but only in the soma, or have other targets entirely.

6. Comparative functional studies

Several labs have investigated the function of homologs of C. elegans sex
determination genes. An early study on C. briggsaetra-1 (de Bono and Hodgkin, 1996) employed partial rescue of C. elegans mutants to demonstrate
conservation of function. However, the discovery of RNAi has had a profound impact.
Kuwabara (1996) first showed the efficacy of RNAi in a non-elegans
species, against Cb-tra-2. Although rescue assays have continued to be used (Chen et
al., 2001; Hansen and Pilgrim, 1998), the phenomenon of
interspecies protein-protein incompatibility discussed above makes partial or failed
interspecies rescue uninformative. Unfortunately, RNAi has its own problems, the
chief being its frequently lower effectiveness in C. briggsae and C. remanei than in
C. elegans (Haag and Kimble, 2000; Haag et al., 2002;
Kuwabara, 1996, M. Montgomery, pers. comm.). For this reason, several
laboratories have begun forward and reverse genetic screens for true sex
determination mutations in C. briggsae (D. Pilgrim, E. Haag, and R. Ellis,
unpublished data). In Pristionchus pacificus, both morpholino oligonucleotides and
forward mutagenesis screens have recently been employed with success (Pires-daSilva
and Sommer, 2004).

Despite these technical difficulties, sex determination function has already been
demonstrated for numerous orthologs of key C. elegans genes. Cb-her-1 is required
for normal male development, and its overexpression clearly masculinizes XX C.
elegans animals. Overexpression of her-1 from the filarial parasite Brugia malayi
was less successful (Streit et al., 1999). tra-2 has conserved roles
in both the soma and germ line of C. briggsae (Kuwabara, 1996) and C.
remanei (Haag and Kimble, 2000), and in vitro and in vivo evidence
exists for its translational repression in these species (Haag and Kimble, 2000; Jan et al., 1997). Similarly, tra-1 performs a key
female-promoting role in other species, both across Caenorhabditis (de Bono and
Hodgkin, 1996) and in the neodiplogasterid Pristionchus pacificus
(Pires-daSilva and Sommer, 2004).

The fem genes of non-elegansCaenorhabditis species have also been studied, with
more surprising results. Although fem-1, fem-2, and fem-3 perform conserved roles in
somatic sex determination, RNAi data (Haag et al., 2002; Stothard et
al., 2002) and two mutations (Hill et al., unpublished
data) indicate that they are dispensable for hermaphrodite spermatogenesis in C.
briggsae. In addition, fem-1 (A. Spence, pers. comm.) and fem-3 (Haag et al., 2002) are probably not required for at least some male spermatogenesis. Despite these
differences, however, the germline-specific fog-1 and fog-3 have conserved roles in
promoting spermatogenesis (Chen et al., 2001). The potential
significance of these results is discussed further below.

7. Mating system evolution in Caenorhabditis

From a phylogenetic perspective, C. elegans is rather unusual in having a
hermaphrodite sex. Its combination with males, termed androdioecy, is found in
animals only in other nematodes and some crustaceans (Sassaman, 1995; Sassaman
and Weeks, 1993). With the exception of C. briggsae, the remaining
Caenorhabditis species are all gonochoristic (male/female). That hermaphroditic
Caenorhabditis are derived from gonochoristic ancestors has been accepted for many years (Honda, 1925), as has the independent origin of selfing in distantly related species (Fitch,
2002). However, recent phylogenies suggest that even C. elegans and C.
briggsae acquired their shared hermaphroditism via parallel evolution (Cho et al.,
2004; Kiontke et al., 2004). An independent origin of
bisexual germline development may help explain discrepancies in germline gene
function that have begun to emerge from comparative functional studies.

In C. elegans, the core somatic pathway is also required for normal germline sex
determination, although its regulation differs from the soma in some details (Figure
3A). First, a direct TRA-1/TRA-2c interaction (Lum et al., 2000; Wang
and Kimble, 2001) is required for reliable hermaphrodite
spermatogenesis. Lum et al. (2000) suggested that this interaction,
while generally female-promoting, is a necessary prerequisite for full repression of
tra-2 that allows initiation of hermaphrodite spermatogenesis. Second, the fem genes
positively regulate spermatogenesis (Hodgkin, 1986) and germline-specific
fog-3 transcription (Chen and Ellis, 2000) in the absence of tra-1.
Finally, tra-1 loss-of-function mutants cannot sustain spermatogenesis, a phenotype
at odds with their complete somatic masculinization (Schedl et al., 1989). This long-puzzling result may stem from both positive and negative
regulation of fog-3 by TRA-1 (Chen and Ellis, 2000).

Figure 3. Models for germline sex determination in C. elegans and C. briggsae. (A) C. elegans, and (B) C. briggsae. In both panels, arrows indicate positive
regulation, and crossbars indicate repressive regulation. With the exception of fog-3,
the genes in black type are also crucial in somatic sex determination. Mechanisms promoting the initiation of
hermaphrodite spermatogenesis are colored green, and those promoting the switch to oogenesis are colored red. Though it is
likely that C. briggsae hermaphroditism is controlled by genes regulating the core pathway downstream of the Cb-fems, their identity is unknown. See text for full explanation.

How much of the regulation described above is used by C. briggsae hermaphrodites?
There is evidence that the repression of tra-2 by a GLD-1-like factor (Jan et al.,
1999) and a TRA-1/TRA-2c direct interaction (Wang and Kimble, 2001) both occur. This is consistent with a central role for tra-2
regulation in the initiation of C. briggsae hermaphrodite spermatogenesis (Figure
3B). However, fog-2, a key cofactor of GLD-1, is unique to C. elegans (see above).
In addition, RNAi directed against the C. briggsaegld-1 ortholog causes highly
penetrant germline masculinization, not the feminization expected from C. elegans
(Nayak et al., 2005). As discussed by Nayak et al. (2005), these latter results suggest several alternative possibilities. One is
that Cb-GLD-1 does not translationally repress Cb-tra-2, or that it does but also
has other, male-promoting targets that are more important. Alternatively, fog-2 may
qualitatively modify the function of Ce-GLD-1 in a conserved GLD-1/tra-2
interaction. It is still possible, however, that the unexpected Cb-gld-1(RNAi)
phenotype is due to incomplete inactivation of Cb-gld-1, as RNAi is generally less
effective in C. briggsae (Haag et al., 2002, M. Montgomery, pers.
comm.). Mutations in Cb-gld-1 will be required to clarify this matter.

In addition to the evolution of the “sperm on” portion of hermaphrodite germline
development, evidence is mounting that the “sperm off” component is also distinct.
While the Cb-fem genes have conserved roles in male somatic development, none have
yet been shown to be required for hermaphrodite spermatogenesis (Haag et al., 2002, A. Spence, pers. comm.; Stothard et al., 2002, Hill et al., unpublished data). Thus
the “sperm off” function must target other parts of the pathway, suggested here
(Figure 3B) to be variously the TRA-1/TRA-2c interaction, direct upregulation of
tra-1, or repression of fog-3. fog-3 expression is tightly associated with sperm
production in both species, as well as in C. remanei (Chen et al., 2001). Surprisingly, Cb-fem-2 and Cb-fem-3 null XO mutants are neither females (Fog), as
would be the case in C. elegans, nor somatic females with spermatogenic germ lines
(Mog), as might be expected from the lack of phenotype in XX hermaphrodites.
Instead, these XO mutants are also hermaphrodites (the Her phenotype; Hill et al., unpublished data). This
suggests that the Cb-fem genes are neither required to initiate nor to curtail hermaphrodite spermatogenesis, but do act to repress the sperm-oocyte
switch in XO males. Overall, it is likely the entire control of hermaphrodite spermatogenesis lies downstream of the fem genes in C. briggsae (Figure 3B).

The question of how hermaphrodites differ from their female ancestors has
motivated much of the comparative work on Caenorhabditis sex determination. C.
briggsae and C. remanei represent the most closely related
gonochoristic/androdioecious species pair (Cho et al., 2004; Kiontke
et al., 2004), and thus make an especially informative comparative
system for future research. More distantly related hermaphrodite species exist that
have convergently evolved selfing (Fitch, 2002). The availability of both
closely related species with different mating systems and multiple cases of parallel
evolution of a complex trait makes soil nematodes an attractive system for
developmental studies of reproductive adaptation. Rapid developments in genome
sequencing and the advent of comparative genetics are making rigorous studies in
these other species possible, and not only for sex determination. However, given the
intrinsic organismal importance and remarkably rapid evolution of sex determination,
it is likely that it will remain a major topic in nematode comparative biology for
the foreseeable future.

8. Acknowledgements

The author thanks Dave Pilgrim, Elizabeth Goodwin, Tim Schedl, Ron
Ellis, Judith Kimble, and Andrew Spence for sharing results prior to publication, and
Barbara Meyer and two anonymous reviewers for helpful comments on an early draft of this
manuscript. Research in the Haag Lab is supported by the National Science Foundation.